The use of microbial products in drug discovery is an ancient and well-established practice. Actinomycetes are known producers of pharmacological and anti-viral agents. This study aimed to screen the crude extract of Actinomycetes some Egyptian collection for their anti (Hepatitis A Virus (HAV-H10), Coxsackie B4 virus (COX B4) and Herpes Simplex Virus (HSV-1)) then, select the most potent actinomycete crude extract (having highest antiviral activity) for processes of purification, identification and mechanism of action. The maximum non toxic dose (MNTD) of each actinomycete extract was determined then antiviral activity against fast growing viral strains replicating in African green monkey kidney (VERO) cells was studied by the reduction in the number of plaques formed by the viruses. A total of 18 extracts Actinomycete isolates with antimicrobial potential against bacteria and fungi was screened for their antiviral activity. The results indicated that; Mnf-21kt extract showed the most promising antiviral activity against three virus strains while extracts from kfs-1ss and kfs-7ss showed inhibition activity against HAV-H10 and HSV-1 only. After purification of extract Mnf-21kt then the analysis of physico-chemical, elemental and spectroscopic analysis (UV, IR, H.NMR, Mass spectroscopy) indicated that; the active compound has the nature of nucleoside analog. On other hand, when the purified active substance was tested for its mechanism of action against HAV-H10, the result indicated that it has induced significant anti-infectivity and anti-replicative effect. The purified compound has promising broad spectrum antiviral activity in an in vitro system.